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16 March 2021 Effects of air pollution on chlorophyll content and morphology of lichens transplanted around a paper industry (Morelia, Mexico)
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Abstract

We evaluated and compared the physiological and morphological response to pollution of two epiphytic lichen species, the foliose lichen Flavopunctelia praesignis and the fruticose lichen Usnea ceratina. Lichen samples were collected at remote areas and transplanted at different distances and directions from a paper industry in Morelia (Michoacán, Mexico). Lichen transplants were exposed for 4 months (1) around the industrial area and (2) in their native habitats (control sites). Changes of total chlorophyll content between samples before and after exposure, and morphological damage in the lichen thalli were investigated. Lichens showed species-specific responses. Flavopunctelia praesignis increased in total chlorophyll content after exposure around the industrial area and in control site. This suggests that total chlorophyll changes by a seasonal effect than pollution effect. On the other hand, chlorophyll content of U. ceratina did not change significantly after exposure. Bleaching, changes in color, deformations, and necrosis of lichen thalli were better visible in U. ceratina near the paper industry. We conclude that U. ceratina is a more pollution-sensitive species than F. praesignis. Morphological damage in the lichen thalli would be a suitable indicator for monitoring early biological effects of air pollution caused by the paper industry.

Lichens are recognized as being very sensitive to air pollution (Munzi et al. 2009). This sensitivity is related to their biology (Nash 2008). Lichens are symbiotic, perennial and slow-growing organisms that depend on wet and dry atmospheric deposition for nutrients (Loppi & Pirintsos 2003). Moreover, the lack of cuticle and stomata allows for many contaminants to be absorbed over the whole lichen surface (Hale 1983). Due to these characteristics, lichens are suitable bioindicators of effects of air pollution, providing reliable information on the quality of the environment (Nimis et al. 2002).

It is known that air pollution has an impact on the morphology and physiology of lichens, affecting the vitality of both mycobiont and photobiont (Garty 2001). The effects of air pollution on lichens include visible morphological changes, i.e., color, form, structure (Estrabou et al. 2004; Fernández-Salegui et al. 2002; Jóźwiak & Jóźwiak 2009; Käffer et al. 2012; Sigal & Nash 1983), ultrastructural changes (Paoli et al. 2015), decreased photosynthesis (Häffner et al. 2001; Picotto et al. 2011), alterations in cell membranes (Paoli et al. 2011), and reduction of photosynthetic pigments (Bajpai & Upreti 2012; Bajpai et al. 2010; Carreras et al. 2009; Garty et al. 2001). These morphological and physiological parameters reflect the health status of lichens and indicate stress caused by air pollution (Lackovičová et al. 2013).

The sensitivity of lichens to air pollution varies among species so that there are sensitive species and tolerant species (Nash 2008). The response of lichens can be influenced by thallus morphology (Escandón et al. 2016; Häffner et al. 2001; Paoli et al. 2015) and ecological characteristics (Bosserman & Hagner 1981; Paoli et al. 2010b; Picotto et al. 2011).

The paper industry is an important source of air pollution (Singh & Chandra 2019). In particular, the mills that use a chemical pulping process (i.e., Kraft) can emit pollutants from chip-storage, digesters, pulp washing, bleaching, recovery of chemicals, evaporators, boilers, lime kilns and dissolving tanks (Surh et al. 2015). These emissions include several airborne pollutants such as SO2, NOx, reduced sulfur compounds, particulate matter, heavy metals, chlorine compounds and organic volatile compounds (Cheremisinoff & Rosenfeld 2010; EPA 2002; Surh et al. 2015). Previous studies on biological effects of air pollution near paper industries have shown the impacts on lichen physiology (Azevedo et al. 2012; Sheridan et al. 1976) and lichen ultrastructure (Holopainen 1983).

In Mexico and other tropical countries, the use of lichens as bioindicators of air pollution is still uncommon (Herrera-Campos et al. 2014). Furthermore, we know little about the suitability of common species as biomonitoring tools (Hawksworth et al. 2005). In the present study, we carried out a biomonitoring study around an industrial area in west central México using common lichen species. Our aim was to evaluate and compare the response to air pollution of two epiphytic lichen species, the lichen foliose Flavopunctelia praesignis and the fruticose lichen Usnea ceratina, transplanted from nearby forests to the vicinity of a pulp and paper industry in Morelia, Mexico. To test whether lichens respond to pollution, we evaluate changes in total chlorophyll content in lichen thalli after exposure, expressed as a percentage change. Moreover, we wanted to evaluate visible morphological damage to the lichen thalli. Finally, we investigated the effect of direction of exposure and distance from the paper industry on lichen response.

In the present work, we hypothesize that: (1) the two lichen species will show different responses to air pollution in the study area due to differences in the thallus morphology (foliose vs. fruticose) and (2) morphological and physiological parameters (pollution damage and chlorophyll content) will vary in relation to direction of exposure and distance from the industry, assuming the effect of topography and prevailing winds in the study area.

The results of this study may help to detect early signs of environmental changes in the study area and also to detect risk areas. Moreover, we can determine the suitability of common lichen species as biomonitors.

Methods

Study area. The study area is located 5 km SW of Morelia city (Michoacán, Mexico) between 19°38′–19°40′N and 101°18′–101°13′W (Fig. 1). The landscape is dominated by agricultural lands and rural areas. The area is crossed by roads and it is located near the highway MEX14. There is a protective site (‘La Mintzita') in the area. Elevations range between 1890 and 2050 m. The climate is temperate sub-humid with summer rains (Fig. 2). The mean annual temperature is 17.8°C, with a minimum of 14.3°C (January) and a maximum of 20.8°C (May). The mean annual precipitation is 816 mm, and it is mainly distributed between May and September. The prevailing winds blow from the SE (data from meteorological station ‘La Mintzita’ OOAPAS 2018).

Figure 1.

Study area with location of the 20 monitoring stations and control sites. 1–5: sites; North: N; East: E; South: S; West: W.

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Figure 2.

Weather data from meteorological station ‘La Mintzita’ in the study area (OOAPAS 2018). A. Mean monthly precipitation (mm) and mean monthly temperature (°C). B. Direction from which the prevailing winds blow. *study period (April to August 2018).

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The facilities of two Kraft pulp mills, operating since 1976, are located at the study area. According to previous data from instrumental monitoring (SUMA 2015), the paper industry in Morelia is responsible for airborne emissions of SO2 (16.89 ton/year), NOx (0.27 ton/year), COV's (0.04 ton/ year), PM10 (1.29 ton/year), and PM2.5 (1.54 ton/ year). Currently, there is no continuous monitoring of air quality in the study area.

Study design and lichen transplants. Twenty monitoring stations around the paper industry were selected (Fig. 1). These stations were distributed in the north, south, east and west directions (five stations in each direction), at approximate distances of 0, 500, 1000, 1500 and 2000 m from the industry. Locations varied by land use (agricultural and rural areas) and topography. At each monitoring station, we selected three ash (Fraxinus uhdei) and willow (Salix babylonica) trees as sources for the transplants.

To evaluate the biological effects of air pollution in the study area, we used two epiphytic lichens, the foliose lichen Flavopunctelia praesignis and the fruticose lichen Usnea ceratina, as biomonitors. We selected these species because they are (i) common in pine-oak forests near Morelia (Gregrorio-Cipriano et al. 2016), and (ii) easy to collect and identify in field. In Mexico, studies have employed species of Usnea and Flavopunctelia as biomonitors of air pollution (Cervantes et al. 2008; Gómez-Peralta & Chávez-Carmona 1995; Zambrano & Nash 2000). In particular, Usnea ceratina was used to evaluate the physiological effect of urban emissions from Mexico City (Zambrano & Nash 2000).

For transplants, we collected samples of Flavopunctelia praesignis and Usnea ceratina from pine-oak forests of two areas far from pollution sources (Fig. 1) in March 2018. Thalli of F. praesignis were collected from the Voluntary Conservation Area ‘El Tocuz’ (19°29′15.29″N, 101°21′29.87″W), whereas thalli of U. ceratina were collected near the rural area Ichaqueo (19°35′5.48″N, 101°7′47.23″W). These sites are situated about 20 and 16 km away from the study area, respectively, and they have similar environmental conditions: mean annual temperature is 15°C, mean annual precipitation is >1000 mm, and the rainy season occurs from May to October (data from the Servicio Meteorológico Nacional CONAGUA 2011). Although both lichen species are common in the two remote areas, their abundances are different (F. praesignis is more abundant in the ‘El Tocuz’, whereas U. ceratina is in Ichaqueo); thus, we decided to collect them separately.

Thalli of both species were collected from oak trees (Quercus sp.) at about 2 m from ground, together with their original substrate, i.e., twig or bark (Giordani et al. 2020). After that, we placed five transplants of each species on each host tree in the study area. Transplants were put in 20 × 20 cm plastic nets with a mesh size of 1 mm2 (Sujetovienė & Galinytė 2016), at 2 m from ground and then exposed in the direction facing the paper industry. In total, we transplanted 15 samples of each species per each monitoring station (n=30). Additionally, we re-transplanted another group of thalli (n=15) to oak trees in the remote areas where lichens were initially collected (Godinho et al. 2004). Thalli of Flavopunctelia praesignis were collected from the ‘El Tocuz' and then transplanted to trees at the ‘El Tocuz’ again; also, thalli of Usnea ceratina were transplanted to trees at Ichaqueo again. Chlorophyll content and morphological damage measured in F. praesignis and U. ceratina thalli transplanted in their native habitat were used as controls. Lichens were exposed for 4 months from April to August 2018. At the end of the exposure period, the lichens were retrieved and transported to the laboratory.

Changes in total chlorophyll content. To determine changes in total chlorophyll content in the lichen thalli, we measured chlorophyll content before and after exposure. Measurements were carried out using a modulated fluorometer CCM 300 (Opti Sciences Inc.). The modulate light was placed on a single peripheral lobe, roughly corresponding to the last year of growth (Nimis et al. 2001), from each sample of Flavopunctelia praesignis, while in Usnea ceratina a single branch from each sample was measured. Four to six samples of each species were measured per station, including the two control sites. Total chlorophyll content was expressed in units of mass per area, mg m–2 (Gitelson et al. 1999).

In order to calculate changes in chlorophyll contents, we used the measured total chlorophyll values before and after exposure. A chlorophyll change percentage (ChlCP, %) was obtained by calculating the difference between the pre-exposure (before) and post-exposure (after) values of each sample and dividing such a difference by pre-exposure (before) values (Eq. 1). Chlorophyll change percentage had positive and negative values, which indicated an increase or a decrease in total chlorophyll content after exposure, respectively.

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Morphological damage in the lichen thalli. To evaluate morphological damage to lichen thalli, we photographed the lichen samples before and after exposure around the paper industry and in the control sites. Sixty specimens of each species (six specimens per each station) were photographed. Additionally, at the end of the exposure, we examined the lichens (previously photographed) in the laboratory under a stereomicroscope. For each sample, we analyzed: changes in thallus color, bleaching, necrosis, loss of the upper cortex, medulla exposure, and growth abnormalities, i.e., deformations of lobes or branches (Estrabou et al. 2004; Fernández-Salegui et al. 2002). Furthermore, we described the location, frequency, and cover percentage of morphological damage. On this basis, we defined interpretative scales of morphological damage for each lichen species (Table 1). These scales consist of four classes that indicate the degree of morphological damage in the lichen thalli.

Table 1.

Interpretative scales of morphological damage for samples of Flavopunctelia praesignis and Usnea ceratina exposed in the study area.

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Morphological damage classes were mapped using a geographical information system QGIS 2.18.6 (QGIS Development Team 2016). In these maps, colors represented the class of morphological damage that corresponded to each monitoring station, while the size of the symbols indicated the percentage of surface of lichens covered by damage.

Statistical analysis. The normality of data was checked by Shapiro Wilk test. Non-parametric Wilcoxon's signed rank test was used to check the significance of differences (p<0.05) in the chlorophyll content between lichen dependent samples before and after exposure. One-way analysis of variance (ANOVA) was used to detect significant differences (p<0.05) in chlorophyll change percentage between sites of exposure (paper industry vs. control), this for each species. To evaluate the effect of distance from the paper industry (0, 500, 1000, 1500, 2000 m) and direction of exposure (N, S, E, W), we used Linear Mixed Models (LMMs) with a nested design. For these models, the chlorophyll change percentage (numeric variable) was used as response. Distance (numeric variable) and direction (categorical variable) and their interaction were used as fixed effects. The nesting of the monitoring stations in the same directions of exposure was considered as a random effect. When a term was not significant (p>0.05), the model was reduced to use only significant terms (p<0.05). All statistical analyzes was performed in R software (R Core Team 2018).

Results

Changes in total chlorophyll content. The chlorophyll change percentage (ChlCP, %) indicated short-term changes in total chlorophyll content for both lichen species (Tables 2 & 3). After the exposure, samples of Flavopunctelia praesignis transplanted around the paper industry increased in total chlorophyll content (ChlCP from +2.73% to +34.69%) in most monitoring stations. At the control site, total chlorophyll content in this species also increased (+20.64%) (Table 2). The increase of total chlorophyll content was significant in the study area (W = 408; z = –7.01; r = 0.70; p <0.0001) and in the control site (W = 0; z = –2.20; r = 0.90; p = 0.03) (Table 4). On the other hand, Usnea ceratina increased (from +3.22% to +14.73%) their total chlorophyll content in 12 stations after exposure, while the remainder (n=8) decreased (from –0.05% to –8.61%). Chlorophyll content increased (+10.65%) in samples of U. ceratina transplanted in the control site (Table 3). In this species, changes in chlorophyll content between samples before and after exposure around the paper industry and in the control were not significant (W = 1774.5; z = –1.87; r = 0.19; p = 0.06 and W = 0; z = –1.82; r = 0.91; p = 0.12, respectively) (Table 4). Chlorophyll content did not show significant differences in the transplants prior to exposure in the study area and in the control sites, nor among lichen species (data not shown).

Table 2.

Total chlorophyll content (mean ± SD, mg m–2) in Flavopunctelia praesignis before and after 4 months of exposure around the paper industry and in the control site (‘El Tocuz'). Changes in chlorophyll between samples before and after exposure are expressed as a chlorophyll change percentage (ChlCP, %). Distances (m) refer to the paper industry. 1–5: sites; North: N; East: E; South: S; West: W.

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Table 3.

Total chlorophyll content (mean ± SD, mg m–2) in Usnea ceratina before and after 4 months of exposure around the paper industry and in the control site (Ichaqueo). Changes in chlorophyll between samples before and after exposure are expressed as a chlorophyll change percentage (ChlCP, %). Distances (m) refer to the paper industry. 1–5: sites; North = N, East = E, South = S, West = W.

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Table 4.

Comparison of total chlorophyll content between Flavopunctelia praesignis and Usnea ceratina exposed around the paper industry and in the control sites. Values followed by different capital letters indicate significant differences (Wilcoxon's test, p <0.05) in chlorophyll content between samples before and after exposure. Values followed by different small letters indicate significant differences (ANOVA, p <0.05) in chlorophyll change percentage (ChlCP, %) between sites of exposure (paper industry vs. control).

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Exposure site (around paper industry vs. control sites) had no effect on total chlorophyll of both lichen species (Table 4; Fig. 3). Chlorophyll change percentages in Flavopunctelia praesignis and Usnea ceratina samples exposed around the paper industry did not statistically differ (F1,104 = 0.95; p = 0.33 and F1,99 = 1.77; p = 0.18, respectively) from those found in the control sites.

Figure 3.

Effect of site (paper industry vs. control) and direction of exposure (N, E, S, W) on chlorophyll change percentage (ChlCP, %) in samples of Flavopunctelia praesignis (A & B) and Usnea ceratina (C & D). Mean ± standard deviation. Values followed by different small letters indicate significant differences (Tukey's test p<0.05) due to the site or direction of exposure. Control: C; Paper industry: Pi. North: N; East: E; South: S; West: W.

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Direction of exposure (N, S, E, W) had a significant effect (p <0.05) on chlorophyll change percentage in Flavopunctelia praesignis (Table 5). The ChlCP (%) was higher in the transplants of F. praesignis exposed to the south than those exposed in other directions, but it was only statistically different (p <0.05) from the east (Fig. 3B). The ChlCP (%) was not related (p >0.05) to the distance from the paper industry and neither was an interaction between factors (Table 5). On the other hand, the distance and direction of exposure had no effect (p>0.05) on chlorophyll change percentage of Usnea ceratina (Table 5; Fig. 3D).

Table 5.

Results of Linear Mixed Models (LMMs) for the effects and interaction of distance from the paper industry (0, 500, 1000, 1500, 2000 m) and direction of exposure (N, E, S, W) on chlorophyll change percentage (ChlCP, %) in Flavopunctelia praesignis and Usnea ceratina around the paper industry. Chi-square value X2 (degrees of freedom). Values in bold indicate the significance (p <0.05) of a factor.

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Morphological damage in the lichen thalli. Both Flavopunctelia praesignis and Usnea ceratina showed visible morphological damage after 4 months of exposure around the paper industry (Figs. 4B,C & 5B,C). The control samples, on the other hand, showed visibly health thalli (Figs. 4A & 5A). According to the damage scale, the two lichen species showed a “normal” condition after exposure in the control sites (Table 1).

Figure 4.

Photographs of Flavopunctelia praesignis after exposure at the control site (‘El Tocuz') and around the paper industry. A. Control. Thallus without visible morphological damage. B. Paper industry. Thallus with reddish-brown dots and bleached areas on the lobe margins (pointed by arrows). C. Detail of apothecia with loss of upper cortex and exposure of the medulla (arrows). Scale bars: 1 cm (A & B); 5 mm (C).

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Figure 5.

Photographs of Usnea ceratina after exposure at the control site (Ichaqueo) and around the paper industry. A. Control. Thallus without visible morphological damage. B. Paper industry. Thallus with necrosis (pointed by arrows). C. Detail of a thallus branch with loss of upper cortex and exposure of the medulla (arrows). Scale bars: 1 cm (A & B); 2 mm (C).

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Flavopunctelia praesignis had thalli from “normal” morphology to thalli with “severe damage” (Table 1; Fig. 6A). Thalli without visible morphological damage were recorded in two stations (10%) located to the south and west from the industry up to 1500 m. Thalli with “little damage” were recorded in five stations (25%) between 1000 and 2000 m, mainly to the east. In these samples, appearance of reddish-brown, rose, and orange small dots and bleached areas were observed. Thalli with “moderate damage” were recorded in eleven stations (55%) located in the north, east, and west directions between 500 and 1000 m. In these stations, reddish-brown dots and bleached areas increased in cover. Finally, samples from two stations (10%) located to the north (<500 m) and south (>1500 m) from the industry, showed thalli with “severe damage”. In these samples, some small areas with exposure of the medulla and necrosis were observed.

Figure 6.

Graphical interpretation of the observed morphological damage in the lichens Flavopunctelia praesignis (A) and Usnea ceratina (B) around the paper industry. Morphological damage classes (see Table 1) are shown by different colors. Symbol sizes indicate the percent cover (%) of damage in each monitoring station. 1-5: sites. North: N; East: E; South: S; West: W.

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Usnea ceratina had thalli from “normal” morphology to thalli with “severe damage” (Table 1; Fig. 6B). Thalli without visible morphological damage were observed only in one station (5%) located to the south from the paper industry up to 2000 m. Samples with “little damage” showed reddish-brown small dots, bleached areas, and recurved branches. These damages were recorded in three stations (15%) located in the north, south, and west directions between 1000 and 2000 m. Thalli with “moderate damage” were observed in eleven stations (55%) at distances from 500 to 2000 m, particularly in lichens exposed to the east and west. In these stations, reddish-brown dots and bleached areas increased in cover. Moreover, loss of upper cortex, exposure of the medulla, and small necrotic areas were observed. Finally, samples from five stations (25%), two from the north, and one from the east, south, and west, showed thalli with “severe damage,” which consist in the detachment of branches and thalli almost completely necrotic. These damages were more visible in the transplants exposed between 0 and 500 m.

At the end of the exposure, morphological damage was more evident in Usnea ceratina than Flavopunctelia praesignis (see percent cover % in Fig. 6A,B). In both species, the percent cover of morphological damage in the lichen thalli increased near paper industry.

Discussion

Lichen species responses. This study shows that the epiphytic lichens Flavopunctelia praesignis and Usnea ceratina have a species-specific response to air pollution in the study area. We found that F. praesignis increased in total chlorophyll content after exposure around the paper industry and in control site, suggesting an apparent enhancement in the performance of the photobiont in this species in both sites of exposure. Morphological damage was less visible and only occasionally observed in samples of F. praesignis. On the other hand, U. ceratina also increased in total chlorophyll after exposure in both sites, although chlorophyll change (ChlCP, %) was lower than in F. praesignis (see Table 4). Further, morphological damage was more visible and frequent in U. ceratina than in F. praesignis.

The differences observed in the responses of the two lichen species to the new ambient conditions may be related to their morphological differences, because thallus morphology may play a role in the resistance of lichens to air pollution (Häffner et al. 2001). According to Bosserman & Hagner (1981), the fruticose thallus of Usnea exposes to the atmosphere more surface area per unit of biomass than does a foliose flattened thallus. In our case, the foliose lichen Flavopunctelia praesignis has a relatively flat thallus which is tightly adnate to its substrate by numerous points of attachment, suggesting less surface area exposed to air pollutants than in U. ceratina.

The species ecology is also important to explain the air pollution tolerance of lichen species (Bosserman & Hagner 1981; Paoli et al. 2010b; Picotto et al. 2011). For instance, Usnea ceratina only occurs on non-eutrophicated barks of Quercus in undisturbed forests, whereas Flavopunctelia praesignis can occur on non to weak eutrophicated barks (i.e., deposition of dust and NOx) in natural, semi-natural habitats and moderately disturbed areas (Nimis & Martellos 2016). On the other hand, it is known that Usnea species are more hygrophytic and more influenced by rainfall elements than Parmelia species such as F. praesignis (Bosserman & Hagner 1981). In the present study, F. praesignis and U. ceratina were collected at undisturbed forests and transplanted under similar conditions; however, the differences in climate with the control sites (temperature of 15°C vs. 17.8°C and precipitation of >1000 mm vs. 816 mm) and a greater deposition of pollutants (i.e., SO2 and NOx) in the study area could affected the performance of more hygrophytic and pollution-sensitive species such as U. ceratina. In contrast, species that are mesophytic and pollution-tolerant such as F. praesignis could show a greater adaptation to environmental conditions.

The responses of fruticose and foliose species have been compared in similar short-term exposures. The fruticose lichen Bryoria fuscescens, exposed at different polluted sites in Germany following an SO2 gradient, showed a decrease of chlorophyll content and in photosynthesis compared to the foliose lichen Parmelia sulcata, which maintained its photosynthetic activity after 4 months exposure (Häffner et al. 2001). Lower chlorophyll content was recorded in the fruticose lichen Evernia prunastri than in the foliose lichen Flavoparmelia caperata after 4 months around an industrial area in Portugal (Godinho et al. 2004). A negative effect of air pollution around a cement mill in Slovakia on photosynthetic performance and ultrastructural alterations were more evident in E. prunastri than in the foliose lichen Xanthoria parietina after 6 months (Paoli et al. 2015).

Chlorophyll content changes. In our study, no negative effects of the pollution source on total chlorophyll were found in Flavopunctelia praesignis. We observed a significant increase in total chlorophyll content in samples of F. praesignis after 4 months of exposure both the paper industry and the control site. The similar behavior among transplanted lichens suggests that observed change in chlorophyll content can be related to factors other than pollution.

Photosynthetic pigments in lichen thalli vary naturally due to the photobiont (Chettri et al. 1998) and climatic conditions (Paoli et al. 2010b). In particular, the chlorophyll content can be strongly affected by seasonal variation (Boonpragob & Nash 1991; Czeczuga & Krukowska 2001). For instance, Boonpragob & Nash (1991) observed lower chlorophyll contents and photosynthetic rates in summer (before precipitation) than in winter (during and after precipitation), suggesting a greater negative effect of air pollutants during dry season, when the lichens are metabolically less active. In our study, we collected and transplanted the lichens before the beginning of the rainy season in April 2018. Later, we retrieved the samples almost at the end of the rainy season in August 2018. Thus, the increase in total chlorophyll in the transplants both in the study area and control site may be due to a seasonal effect than a pollution effect.

Alternatively, an increase in the chlorophyll synthesis in the samples of Flavopunctelia praesignis exposed around the paper industry could also be stimulated by atmospheric pollutants. Low levels of pollutants may have a fertilizing effect on photosynthetic pigments (Ochoa-Hueso & Manrique 2011; Ra et al. 2005; Sujetovienė & Sliumpaitė 2013; Wakefield et al. 2011). According to Ra et al. (2005), low concentrations of sulfur and nitrogen compounds can be used as nutrients by some air pollution-tolerant lichens and enhance metabolic performance, resulting in higher levels of chlorophyll content. On the other hand, atmospheric pollutants can be responsible for both the pigment degradation and the increase of its synthesis (Carreras et al. 2005, 2009). For instance, samples of Usnea sp. showed an increased in the degradation of chlorophyll a, and at the same time, an increase in total chlorophyll content parallel to levels of pollutants emitted by vehicular traffic in city of Córdoba, Argentina (Carreras et al. 1998). They proposed chlorophyll synthesis was a compensatory mechanism for negative effects of air pollution.

On the other hand, the total chlorophyll in Usnea ceratina seems not to be influenced by time and the transplantation site, as there was no significant difference in chlorophyll content between before and after exposure or between transplanted lichens around the paper industry and in the control site. The fact that the chlorophyll of this species was not sensitive to pollution could be due the duration of exposure. After longer exposures, the lichens became saturated with elements, lose biomass, and alter surface structures and physiological performance (Bargagli & Mikhailova 2002). In the present study, we observed severe morphological damage in U. ceratina after 4 months exposure around the industry, which could affect the performance of this sensitive-pollution species during the study period. The sensitivity of Usnea species to contamination has been reported (Carreras et al. 2005; Escandón et al. 2016; Hawksworth & Rose 1970; Zambrano & Nash 2000).

Parameters such as chlorophyll degradation, chlorophyll a/b ratio, photosynthetic rates and chlorophyll fluorescence can detect responses earlier than for total chlorophyll content (Bačkor & Dzubaj 2004; Bačkor et al. 2007; Chettri et al. 1998; Häffner et al. 2001; Picotto et al. 2011). On the other hand, several studies recognized that cell membrane damage (Munzi et al. 2009; Paoli & Loppi 2008; Paoli et al. 2011; Sujetovienė & Galinytė 2016) or changes of the respiration rates (Paoli et al. 2015) in the mycobiont are better indicators of pollution stress symptoms than photosynthetic parameters. In the present study, we did not detect a stress response (i.e., reduction of photosynthetic pigments) on the photobiont of both lichen species at the industrial site indicated by chlorophyll content. Therefore, the use of more sensitive ecophysiological parameters of the photobiont and the mycobiont (i.e., photosynthesis, chlorophyll fluorescence, and cell membrane damage) could help us measure early biological effects of air pollution.

Adverse effects of pollutants also depend on exposure duration (Paoli et al. 2010a). Häffner et al. (2001) reported the loss of chlorophyll content with an increase of levels of SO2 in the pollution-sensitive species Bryoria fuscescens within 4–8 weeks of exposure. In contrast, other studies (Paoli & Loppi 2008; Paoli et al. 2011) suggest that short exposure times of 1–3 months may not be sufficient to observe alterations on the photosynthetic performance. In our study, Flavopunctelia praesignis and Usnea ceratina were exposed for 4 months and it cannot be excluded that different exposure times would be necessary to detect stress responses in both lichen species.

Morphological damage in the lichen thalli. Both Flavopunctelia praesignis and Usnea ceratina showed visible morphological damage after 4 months of exposure around the paper industry. At the control sites, on the other hand, the two species did not show morphological damage between time periods. This latter result confirmed that our transplant itself does not affect the lichen responses, and therefore the observed damage must reflect the influence of environmental conditions. In the same way, the morphological damage in the lichen thalli seem unrelated to seasonal variations.

In the present study, the observed morphological damage, i.e., brown-reddish spots, bleaching, growth abnormalities, loss of upper cortex, exposure of the medulla, and necrosis were more evident in Usnea ceratina than Flavopunctelia praesignis. In U. ceratina, the percent cover of damage in lichen thalli ranged from 20 up to 80%. Several damages were observed in the fruticose lichen Ramalina celastri, a sensitive species, compared with tolerant and foliose species Physcia endochrysea and P. undulata (Estrabou et al. 2004).

The presence of brown-reddish spots on lichen thalli have been associated with the degradation of chlorophyll to phaeophytin (brown pigment) under acidic conditions derive from the exposure to SO2 (Rao & LeBlanc 1966). This damage was commonly observed in the lichens transplanted in the study area and it is consistent with reports from several lichen species exposed to high levels of SO2 (Fernández-Salegui et al. 2002; Gómez Peralta & Gómez-Reyes 2007; Häffner et al. 2001). The visible bleaching of the lichen thalli is caused by the breakdown or loss of chlorophyll (Godinho et al. 2004). Bleached patches have been reported in lichens at sites contaminated by SO2 (Gómez Peralta & Gómez-Reyes 2007; Häffner et al. 2001; Paoli & Loppi 2008), volatile organic compounds (Wakefield et al. 2011), metals (Käffer et al. 2012), and oxidant pollutants (Sigal & Nash 1983). In our study, the bleaching of thalli was frequently observed in the samples of both lichen species.

Growth abnormalities, i.e., convoluted lobes or recurved branches, were observed in both species, although this response was better visible in Usnea ceratina. In general, the two species showed more compact thalli after exposure. Similar observations were made in the lichens Parmelia sulcata exposed to SO2 (Fernández-Salegui et al. 2002; LeBlanc & Rao 1973), Hypogymnia enteromorpha exposed to oxidant pollutants (Sigal & Nash 1983), and in several species of Usnea (Otnyukova 2007) as a stress response caused by metal pollution.

Other visible signs of pollution damage in transplanted lichens were the disintegration or loss of the upper cortex (included the algal layer) and exposure of the medulla. These damages were also found in samples of Parmotrema tinctorum at polluted sites with high levels of NOX, SO2 and O3 in Japan (Ohmura et al. 2009). The same responses were reported from different lichen species (Fernández-Salegui et al. 2002; Gómez-Peralta & Gómez-Reyes 2007). In the present study, we believe that the loss of the upper cortex could indicate pollution damage to the mycobiont since the fungal hyphae constitute the upper layer of lichen thalli, which is in direct contact with the polluted atmosphere.

Large areas of necrosis on lichen surface were observed in Usnea ceratina, while in Flavopunctelia praesignis small necrotic patches were occasionally observed. Necrotic thalli were also observed in lichens exposed to SO2 and particulate matter in urban (Estrabou et al. 2004; Glenn et al. 1995; Gómez-Peralta & Gómez-Reyes 2007) and industrial sites (Fernández-Salegui et al. 2002; Gómez-Peralta & Chávez-Carmona 1995), and heavy metals (Jóźwiak & Jóźwiak 2009; Käffer et al. 2012).

The morphological damage in Flavopunctelia praesignis and Usnea ceratina thalli could be related to SO2. It is widely assumed that SO2 is very toxic to lichens (Nash 2008). This gas is very soluble and can dissolve in rainwater (forming acid rain) or in moisture within the cell walls of wet lichen thalli (Richardson 1992). Under aqueous conditions, SO2 dissociates to more reactive forms (i.e., H2SO4 or HSO3) and can disrupt the photosynthesis, respiration and the integrity of cell membrane (Conti & Cecchetti 2001). In our study area, one of main sources of SO2 is the paper industry. SO2 and other sulfur compounds (i.e., H2S and mercaptans) are mainly originate in the black liquor during the recovery boiler operations (Surh et al. 2015). In Morelia, 43% emissions of SO2 derive from pulp and paper industry and 9.3% from vehicular emissions (SUMA 2015).

Effect of distance and direction of lichen response. Our results indicate that the direction of exposure affects the response of Flavopunctelia praesignis. We found that total chlorophyll content and chlorophyll change percentage were higher in the south than other directions, while the lowest chlorophyll contents were observed in the transplants exposed to the east. Morphological damage and percent cover did not show a clear pattern; however, samples of F. praesignis with “moderate” to “severe” damage and higher percent cover of damage were recorded in the north and west directions. On the other hand, we did not observe a significant effect of the direction of exposure on chlorophyll of Usnea ceratina. Nevertheless, this species showed greater pollution damage to the north and west from the industry (see percent cover Fig. 6B).

The variations in chlorophyll content and thallus morphology of both lichen species regarding the direction of exposure are consistent with the direction from which the prevailing winds blow in the study area. Sites to the north and west, which are in the direction of prevailing winds (from SE to NW in our case), were the most influenced by urban and industrial emissions, showing lower chlorophyll contents and higher cover of damage in both lichen species after exposure. Near these sites, we found rural areas, agricultural lands, and the protected site'La Mintzita' that risk high levels of contamination.

The total chlorophyll content was not significantly related to distance from the source. This result may be explained by the fact that the distances between monitoring stations are not great enough to show the effect of the proximity of each station to the paper industry on chlorophyll. However, the two lichen species displayed greater signs of pollution damage at 500 and 1000 m from the paper industry, which suggests that the air emissions from this industry could be related to the observed morphological damage. In this study, therefore, we conclude that chlorophyll content is not a good indicator of damage in lichens while morphological damage was a more sensitive parameter to assess the effect of air pollution from the industry on the vitality of both lichen species.

On the other hand, the degree of morphological damage in the samples of Usnea ceratina confirmed that this species is more sensitive to air pollution than Flavopunctelia praesignis. As a consequence, this study underlines the utility of thallus morphology in biomonitoring studies. Finally, the present work provides baseline data on physiological and morphological response of two lichen species commonly distributed in Mexico, which will be helpful for carrying out future biomonitoring studies in the same area or in other tropical regions.

Acknowledgments

The authors express particular thanks to Arnulfo García Blanco for facilitating the use of the fluorometer for chlorophyll measurements. The Laboratorio de Macromicetes y Liquenes and the Voluntary Conservation Area ‘El Tocuz’ is thanked for their help in the collection and transplantation of samples. Violeta Cortéz Hernández is thanked for the figure edition. Thanks to the Mexican Consejo Nacional de Ciencia y Tecnología (CONACyT) for the scholarship assigned to the first author.

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Copyright ©2021 by The American Bryological and Lichenological Society, Inc.
Violeta Rangel-Osornio, Ana Belén Fernández-Salegui, Víctor Manuel Gómez-Reyes, Ruth Alfaro Cuevas-Villanueva, and Leonel Lopez-Toledo "Effects of air pollution on chlorophyll content and morphology of lichens transplanted around a paper industry (Morelia, Mexico)," The Bryologist 124(1), 52-67, (16 March 2021). https://doi.org/10.1639/0007-2745-124.1.052
Received: 4 June 2020; Accepted: 22 December 2020; Published: 16 March 2021
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